Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.
Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.
复制标题
用于检测番茄植株和粉虱中番茄黄曲叶病毒的高灵敏血清学方法
DOI：
10.1186/1743-422x-10-142
复制
发表时间：
2013-05-06
期刊：
影响因子：
4.8
通讯作者：
Wu J
中科院分区：
文献类型：
--
作者：
Xie Y;Jiao X;Zhou X;Liu H;Ni Y;Wu J
番茄黄卷叶病毒（TYLCV）是双病毒科begomvirus属的一种病毒，对热带和亚热带地区的番茄生产造成严重损失。纯化的TYLCV病毒粒子作为免疫原，利用杂交瘤技术制备单克隆抗体（mab）。建立了基于单克隆抗体的斑点酶联免疫吸附法（dot- elisa）和直接组织印迹免疫法（DTBIA），用于快速、灵敏、简便地检测中国大田番茄和烟粉虱（bmisia tabaci）样品中的TYLCV。采用杂交瘤技术，制备了6种抗TYLCV的小鼠单克隆抗体（1C4、8D10、6E3、2F2、3F4和4G3）。采用单克隆抗体（MAb） 1C4，建立了斑点酶联免疫吸附法（dot-ELISA）和DTBIA检测中国大田番茄和白蝇中TYLCV的方法。dot-ELISA分别在1:5,120 （w/v, g mL-1）稀释的染病组织粗提物和1:128（白蝇个体/μL）稀释的毒粉虱匀浆中检测到TYLCV。采用上述两种方法对来自全国TYLCV流行地区的田间番茄（n=487）和粉虱（n=110）样本进行TYLCV检测，并通过PCR和核苷酸测序对结果进行进一步验证。调查显示，TYLCV在中国浙江、山东和河南等省的番茄上广泛存在。所建立的dot-ELISA法非常适合大田番茄和粉虱样品中TYLCV的常规检测，而DTBIA法更适合于在TYLCV流行地区的大规模番茄植株样品中进行TYLCV的常规检测。
Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus in the family Geminiviridae, which causes severe losses in tomato production in tropic and subtropic regions. The purified TYLCV virions were used as the immunogen to produce monoclonal antibodies (MAbs) using the hybridoma technology. MAb-based dot enzyme-linked immunosorbent assay (dot-ELISA) and direct tissue blot immunoassay (DTBIA) were developed for sensitive, simple, and rapid detection of TYLCV in field tomato and whitefly (Bemisia tabaci) samples collected from TYLCV prevalent provinces in China. Using the hybridoma technology, six murine MAbs (1C4, 8D10, 6E3, 2F2, 3F4 and 4G3) against TYLCV were prepared. Using the MAb 1C4, dot-ELISA and DTBIA were then established for detecting TYLCV in field tomato and whitefly samples collected from TYLCV prevalent provinces in China. The dot-ELISA could detect TYLCV in infected tissue crude extract diluted at 1:5,120 (w/v, g mL-1), and in viruliferous whitefly homogenate diluted at 1:128 (individual whitefly/μL), respectively. Field tomato samples (n=487) and whitefly samples (n=110) from TYLCV prevalent districts in China were screened for the presence of TYLCV using the two developed methods, and the results were further confirmed by PCR and nucleotide sequencing. The survey revealed that TYLCV is widespread on tomato plants in Zhejiang, Shandong and Henan provinces in China. The developed dot-ELISA is very suitable for the routine detection of TYLCV in field tomato and whitefly samples, and the DTBIA is more suitable for the routine detection of TYLCV in large-scale tomato plant samples collected from TYLCV prevalent areas.